Large scale production of d-lactate dehydrogenase for the stereospecific reduction of pyruvate and phenylpyruvate

Abstract

To initiate studies of the stereospecific reduction of pyruvate and phenylpyruvate to the corresponding d-2-hydroxyacids a limited screening was carried out for microorganisms possessing a high NADH-dependet d-lactate dehydrogenase activity. Lactobacillus confusus was found to produce the desired dehydrogenase, which showed also relatively high activity towards phenylpyruvate, so this strain was selected for large scale production of the enzyme. A procedure for large scale purification of the enzyme starting with 24 kg wet cells is described including liquid-liquid extraction, ultrafiltration and chromatography on DEAE-cellulose, yielding a catalyst with specific activities of 216 U×mg−1 for pyruvate reduction and 15 U×mg−1 for phenyl-pyruvate reduction. A further tenfold purification can be achieved by affinity chromatography on Blue-Sepharose C-6B. Parameters which are important for industrial application of the enzyme were determined: substrate specifity, pH and temperature optimum, temperature stability, stability at different pH-values, and the storage stability of the enzyme in crude extracts.